Cyclic  citrullinated  peptide,  rheumatoid  arthritis  diagnosis composition  including  the  same, rheumatoid  arthritis  diagnosis  method using  the   peptide  or  the  composition, and  method  of  screening  diagnostic marker  for  rheumatoid  arthritis

ABSTRACT

Rheumatoid arthritis is efficiently diagnosed with improved patient&#39;s convenience using a rheumatoid arthritis diagnosis composition and a kit, each including cyclic citrullinated peptide (CCP), a rheumatoid arthritis diagnosis method using the CCP, the rheumatoid arthritis diagnosis composition, or the kit, a method of obtaining information for rheumatoid arthritis diagnosis, and a method of screening a novel diagnostic marker for rheumatoid arthritis.

RELATED APPLICATIONS

This application claims the benefit of Korean Patent Application No.10-2015-0042566, filed on Mar. 26, 2015, in the Korean IntellectualProperty Office, the disclosure of which is incorporated herein in itsentirety by reference.

BACKGROUND

1. Field

One or more embodiments of the present invention relate to a cycliccitrullinated peptide (CCP), a rheumatoid arthritis diagnosiscomposition and kit each including the CCP, a rheumatoid arthritisdiagnosis method using the CCP, the rheumatoid arthritis diagnosiscomposition or kit, and a method of screening a diagnostic marker forrheumatoid arthritis.

2. Description of the Related Art

Rheumatoid arthritis (“RA”) is a chronic, inflammatory disease that mayoccur due to autoimmunization of unknown cause. The onset of RA maycause inflammation and pain in the intraarticular synovium (synovialmembrane). Sustained pain from RA may make the inflammatory synovialtissue grow into bone and cartilage, and thus may lead to articulardeformation and finally behavior disorder. Although no drug for completecure of RA is available yet, neglecting such symptoms of RA for a longtime since its onset may cause articular deformation and spread ofinflammation that may damage other organs. Accordingly, it is verycrucial to early diagnose and treat RA.

Since an accurate diagnostic method for RA is not currently available,diagnosis for RA is based on a comprehensive analysis of pathognomonicsymptoms, test results, and the like. However, systemic lupuserythemathode (SLE), arthralgia, or osteoarthritis (OA) patients withsimilar symptoms to RA may be confused with early-stage RA patients, andaccurate diagnosis for RA is still difficult.

Currently available diagnostic markers for RA include a rheumatoidfactor (RF) and an anti-CCP antibody. However, these diagnostic markershave a lower specificity with respect to non-rheumatoid arthritispatients than to normal patients. Therefore, there are needs to discovernovel RA diagnostic markers with a high sensitivity and a highspecificity with respect to non-rheumatoid arthritis and to develop adiagnostic method for RA by using such novel diagnostic markers.

SUMMARY

One or more embodiments of the present invention include a cycliccitrullinated peptide (CCP).

One or more embodiments of the present invention include a rheumatoidarthritis (RA) diagnosis composition including the CCP.

One or more embodiments of the present invention include an anti-CCPantibody detection method for diagnosis of rheumatoid arthritis (RA) ina subject.

One or more embodiments of the present invention include a method ofscreening a diagnostic marker for RA.

Additional aspects will be set forth in part in the description whichfollows and, in part, will be apparent from the description, or may belearned by practice of the presented embodiments.

According to one or more embodiments of the present invention, there isprovided a peptide having an amino acid sequence with a sequenceidentity of about 80% or more to at least one of amino acid sequences ofSEQ ID NOS: 3 to 10.

According to one or more embodiments of the present invention, arheumatoid arthritis diagnosis composition includes a peptide having anamino acid sequence with a sequence identity of about 80% or more to atleast one of amino acid sequences of SEQ ID NOs: 3 to 10.

According to one or more embodiments of the present invention, a methodof detecting an anti-CCP antibody includes: forming an anti-CCPantibody-peptide complex by contacting a sample taken from a subject anda peptide having an amino acid sequence with a sequence identity ofabout 80% or more to at least one of amino acid sequences of SEQ ID NOs:3 to 10 to bind an anti-cyclic citrullinated peptide (CCP) antibodypresent in the sample to the peptide; and detecting a level of theanti-CCP antibody-peptide complex in the sample.

According to one or more embodiments of the present invention, a methodof screening a diagnostic marker for rheumatoid arthritis includes:modifying at least one amino acid of a cyclic citrullinated peptide(CCP) present between citrulline and disulfide bond-forming cycteine ina C-terminal direction from the citrulline; forming anti-CCPantibody-peptide complexes by bringing the modified CCP to contact atest sample taken from a rheumatoid arthritis patient and a controlgroup sample to bind the modified CCP to anti-CCP antibodies present inthe test sample and the control sample group; measuring levels of theanti-CCP antibodies in the anti-CCP antibody-peptide complexes in thetest sample and the control group sample; and determining the CCP as acandidate diagnostic marker for rheumatoid arthritis if the level of theanti-CCP antibody in the anti-CCP antibody-peptide complex in the testsample is higher than that in the control group sample.

BRIEF DESCRIPTION OF THE DRAWINGS

These and/or other aspects will become apparent and more readilyappreciated from the following description of the embodiments, taken inconjunction with the accompanying drawings in which:

FIGS. 1A to 1F are graphs illustrating the autoimmune antibody levels inblood of the normal patients, osteoarthritis patients, systemic lupuserythemathode (SLE) patients, and rheumatoid arthritis (RA) patients, asresults of assay using cyclic citrullinated peptide (CCP, FIG. 1A), arheumatoid factor (RF, FIG. 1B), and modified CCPs without (FIG. 1C) orwith aminocaproic acid (FIGS. 1D to 1F);

FIGS. 2A and 2B are graphs of sensitivity and specificity, respectively,of RF, CCP, and modified CCPs with respect to arthritis;

FIGS. 3A to 3E are graphs illustrating the blood autoimmune antibodylevels in arthritis patients and normal patients detected usingC-terminal sequence-modified CCPs, as a result of enzyme linkedimmunosorbent assay (ELISA);

FIGS. 4A, 4B, and 4C are receiver operating characteristic (ROC) curvesof CCP, and two modified CCPs, respectively, in RA patient samples withrespect to normal patient samples; and FIGS. 4D, 4E, and 4F are ROCcurves of CCP and two modified CCPs, respectively, in RA patient sampleswith respect to osteoarthritis (OA) patient samples; and

FIGS. 5A and 5B are graphs illustrating sensitivity and specificity ofthe tests using CCP and two modified CCPs, and statistical significancesthereof in the diagnosis for RA with respect to normal (FIG. 5A) and OApatient samples (FIG. 5B) as controls.

DETAILED DESCRIPTION

Reference will now be made in detail to embodiments, examples of whichare illustrated in the accompanying drawings, wherein like referencenumerals refer to like elements throughout. In this regard, the presentembodiments may have different forms and should not be construed asbeing limited to the descriptions set forth herein. Accordingly, theembodiments are merely described below, by referring to the figures, toexplain aspects of the present description. As used herein, the term“and/or” includes any and all combinations of one or more of theassociated listed items. Expressions such as “at least one of,” whenpreceding a list of elements, modify the entire list of elements and donot modify the individual elements of the list.

As used herein, the term “amino acid” may include the 22 standard aminoacids naturally incorporated into peptides, and D-isomers and modifiedamino acids thereof.

The modified amino acids may be non-standard amino acids produced bypost-translational modification. Examples of post-translationalmodification include phosphorylation, glycosylation, acylation(including, for example, acetylation, myristoylation, andpalmitoylation), alkylation, carboxylation, ketonization, hydroxylation,glycosylation reactions, biotinylation, ubiquitinylation, a modificationof the chemical properties (for example, beta-elimination, deimination,deamination), a structural modification (for example, formation ofdisulfide bridges), and modification of an amino acid resulting from thechemical reaction of binding with a cross-linking agent to form apeptide conjugate (for example, a modification of an amino group, suchas change in amino group, carboxyl group or side chains)

According to an aspect of the present disclosure, there is provided acyclic citrullinated peptide (CCP).

The term “CCP” as acronym for “cyclic citrullinated peptide” may referto a peptide including citrulline in a cyclic structure with twocysteines linked by a disulfide bond therein.

Citrulline may be an amino acid resulting from post-translationalmodification of a ketimine group of arginine into a ketone group bypeptidylarginine deiminase (PAD).

Peptidylarginine deiminase (PAD) as a hydrolase may hydrolyze a C—H bondof an amidine group.

For example, the peptidylarginine deiminase (PAD) may be PADI1, PADI2,PADI3, or PADI4. The peptidylarginine deiminase (PAD) may be a proteinwith one of SEQ ID NOS: 11 to 14.

The CCP may have an amino acid sequence with a sequence identity ofabout 60% or more to at least one of amino acid sequences of SEQ ID NOs:1 and 3 to 10, for example, about 70% or more, about 80% or more, about90% or more, about 91% or more, about 92% or more, about 93% or more,about 94% or more, about 95% or more, about 96% or more, about 97% ormore, about 98% or more, about 99% or more, or about 100%. In someembodiments, the CCP may be a peptide having an amino acid sequence ofone of SEQ ID NOS: 1 and 3 to 10 in which at least one, at least two, atleast three, at least four, at least five, at least six, or at leastseven amino acids have modified sequences.

As used herein, the term “sequence identity” of a peptide or amino acidsequence used herein refers to a degree of identity of amino acidresidues of two corresponding sequences over a particular regionmeasured after the sequences are aligned to be matched with each otheras much as possible. The sequence identity is a value that is measuredby comparing optimally aligned two corresponding sequences of aparticular comparable region, wherein in the comparable region, a partof the sequence may be added or deleted compared to a referencesequence. In some embodiments, a percentage of the sequence identity maybe calculated by comparing two optimally aligned corresponding sequencesin an entire comparable region, determining the number of locationswhere an amino acid or a nucleic acid is identical in the two sequencesto obtain the number of matched locations, dividing the number of thematched locations by the total number (that is, a range size) of alllocations within a comparable range, and multiplying the result by 100to obtain a percentage of the sequence identity. The percent of thesequence identity may be determined by using known sequence comparisonprograms, for example, BLASTN (NCBI), CLC MAIN WORKBENCH (CLC BIO), orMegAlign™ (DNASTAR INC.).

The peptide may be a wild type peptide identified and separated fromnaturally occurring sources. The peptide CCP according to anyembodiments of the present disclosure may be an artificial variantincluding at least one amino acid having a sequence that is substituted,deleted, and/or inserted compared to at least one of the peptidesincluding amino acid sequences of SEQ ID NOs: 1 to 10. Modification inamino acid of a wild type polypeptide or an artificial variant mayinclude conservative amino acid substitution that does not significantlyaffect folding and/or activity of a protein. Examples of conservativeamino acid substitution may include substitution of a basic amino acid,an acidic amino acid, a polar amino acid, a hydrophobic amino acid, anaromatic amino acid, and a smaller amino acid. The basic amino acid maybe arginine, lysine, or histidine. The acidic amino acid may be glutamicacid or aspartic acid. The polar amino acid may be glutamine orasparagine. The hydrophobic amino acid may be leucine, isoleucine,valine, or methionine. The aromatic amino acid may be phenylalanine,tryptophan, or tyrosine. The smaller amino acid may be glycine, alanine,serine, or threonine. Amino acid substitutions that may lead to nochange in a specific activity are known in the art.

Another type of amino acid variant in peptide may be an amino acidvariant that may result from a modification in a glycosylation patternof an amino acid. The “modification” may refer to deletion of at leastone carbohydrate moiety found in the peptide and/or addition of at leastglycosylation site not present in the peptide.

Typically, glycosylation of peptide may be N-linked glycosylation orO-linked glycosylation. N-linked glycosylation may refer to theattachment of carbohydrate residues to a side chain of asparaginesresidue. Tripeptide sequences asparagine-X-serine andasparagine-X-threonine, where X is any amino acid except proline, arethe recognition sequences for enzymatic attachment of carbohydratemoiety to the asparagines side chain. Accordingly, the presence of apolypeptide with at least one of the tripeptide sequences in apolypeptide may provide a potential glycosylation site. O-linkedglycosylation may refer to the attachment of one of sugarN-acetylgalactosamine, galactose, and xylose to hydroxyamino acid,serine, threonine, 5-hydroxyproline, or 5-hydroxylysine.

Addition of a glycosylation site, for example, an N-linked glycosylationsite, to a peptide may be implemented by modifying the amino acidsequence of the peptide to include at least one of the above-mentionedtripeptide sequences. Addition of an 0-linked glycosylation site may beimplemented by adding at least one serine or threonine residue into thesequence of an original peptide or substituting with at least one serineor threonine residue.

The CCP may be detectably labeled. The labeling may be optical labeling,electrical labeling, radioactive labeling, enzymatic labeling, or anycombination thereof. The optical labeling may be implemented by using afluorescent or phosphorescent substance. Examples of the fluorescentsubstance are fluorescein, rhodamine, cyanine (Cy), metalloporphyrincomplex, Cy-5, and Cy-3. Examples of the fluorescein dye include6-carboxyfluorescein (6-FAM), 2′,4′,1,4,-tetrachlorofluorescein (TET)2′,4′,5′,7′,1,4-hexachlorofluorescein (HEX), 2′,7′dimethoxy-4′,5′-dichloro-6-carboxyrhodamine (JOE),2′-chloro-5′-fluoro-7′,8′-fusedphenyl-1,4-dichloro-6-carboxyfluorescein, and2′-chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein. The enzyme usedin enzymatic labeling may convert a substrate into chromophoricsubstance).

According to another aspect of the present disclosure, there is provideda rheumatoid arthritis diagnosis composition including a CCP accordingto any of the embodiments as described above.

The rheumatoid arthritis diagnosis composition may further include amaterial required for analysis, for example, detection or quantificationof a target material. For example, the material required for analysismay be an antibody or antigen-binding fragment, a cell-staining reagent,a buffer, or the like.

The rheumatoid arthritis diagnosis composition may further a substancethat specifically binds to at least one different rheumatoid arthritismarker, a fragment thereof, or any substance to measure or detect thebinding. The at least one different rheumatoid arthritis marker mayinclude a rheumatoid factor (RF), an anti-CCP antibody, a C-reactiveprotein (CRP), a material associated with inflammation reaction, or anycombination thereof.

As used herein, the term “marker” may refer to any biological index forobjective measurement and evaluation of normal biological processes,disease progression, and reactivity of drugs to treatment. For example,the marker may include blood pressure, body temperature, blood glucoselevel, the presence of gene or generic variants, and the levels ofnucleic acid, protein, peptide, bacteria, or virus.

The term “rheumatoid factor (RF)” may refer to an autoimmune antibodyobserved in a rheumatoid arthritis patient.

The term “C-reactive protein (CRP)” may refer to a protein used todetermine the prognosis of inflammation due to its level increase duringinflammation reaction.

The erythrocyte sedimentation rate (ESR) may be measured to detect amaterial related with inflammation reaction. The ESR refers to the rateat which red blood cells in the venous blood treated with ananticoagulating agent are separated and precipitate from plasma. The ESRmay be increased with the onset of inflammation disease due to anincreased level of such as fibrinogen that is able to bind to red bloodcells, and thus may be used as a marker for inflammation diseases suchas rheumatoid arthritis.

The rheumatoid arthritis diagnosis composition may be in any state, forexample, liquid, solid, or a combination of these states.

The subject to be diagnosed may be a mammal. For example, the mammal maybe a human, a mouse, a rat, a cow, a goat, a pig, a horse, a sheep, adog, a cat, or a combination thereof.

According to another aspect of the present disclosure, there is provideda kit for diagnosing rheumatoid arthritis, the kit including a CCPaccording to any of the embodiments as described above. The kit mayinclude any material that is included in a rheumatoid arthritisdiagnosis composition according to any of the above-describedembodiments. The kit may include a manual in which processes of usingthe components of the kit to diagnose rheumatoid arthritis aredescribed. The kit may further include a reagent for diagnosingrheumatoid arthritis in a subject. For example, the reagent may includea buffer, an indicator, or a combination thereof.

According to another aspect of the present disclosure, there is provideda method of detecting an anti-cyclic citrullinated peptide (anti-CCP)antibody, the method including contacting a test sample taken from asubject and a CCP according to any of the above-described embodiments tobind an anti-CCP antibody present in the sample to the CCP, therebyforming an anti-CCP antibody-CCP complex.

The antibody may be an antibody able to bind to a peptide having anamino acid sequence with a sequence identity of about 60% or more to atleast one of amino acid sequences of SEQ ID NOS: 1 and 3 to 10, forexample, about 70% or more, about 80% or more, about 90% or more, about91% or more, about 92% or more, about 93% or more, about 94% or more,about 95% or more, about 96% or more, about 97% or more, about 98% ormore, about 99% or more, or about 100%.

The method may include contacting a sample taken from a subject and aCCP according to any of the above-described embodiments to bind ananti-CCP antibody present in the sample with the CCP. The contacting maybe performed in a liquid medium. The liquid medium may be a sampleitself in a liquid state, water, a buffer, or a combination thereof. Thecontacting may be performed by mixing the sample with the CCP. Forexample, the contacting may be performed by stirring the sample and theCCP in a container.

The subject may be the same as that described above in conjunction withthe rheumatoid arthritis diagnosis composition according to anembodiment of the present disclosure.

The sample may be a biological material derived from the subject. Thebiological sample may be a solid tissue obtained from a fresh or storedorgan, tissue, or a biopsy; blood or blood components; bodily fluids,such as amniotic fluid, peritoneal fluid, or interstitial fluid; cells;or any combinations thereof. The sample may include a compound naturallyoccurring without being mixed with a biological material such aspreservatives, anticoagulants, buffers, fixatives, nutrients, andantibiotics. For example, the sample may be urine, mucus, saliva, tears,blood plasma, blood serum, sputum, spinal fluid, serous fluid from apleural cavity, nipple aspirate, lymph, tracheolar fluid, intestinaljuice, genitourinary tract fluid, breast milk, semen, peritoneal fluid,cystic tumor fluid, amniotic fluid, or any combinations thereof.

The CCP may be detectably labeled.

In the anti-CCP antibody detection method, the CCP and the labeling maybe the same as described above in conjunction with the composition orkit for diagnosing rheumatoid arthritis.

The anti-CCP antibody detection method may include measuring a level ofthe anti-CCP antibody present in the sample. The measuring of the levelof the anti-CCP antibody may include directly or indirectly measuringthe level of the anti-CCP antibody, for example, by measuring thepresence or the amount of the CCP and/or an anti-CCP antibody-CCPcomplex. In this case, the CCP may be labeled, so that the level of theanti-CCP antibody may be measured by detecting a signal from thelabeling of the CCP.

The measuring of the level of the CCP and/or the anti-CCP antibody-CCPcomplex may include measuring the amount of the CCP after separationfrom the anti-CCP antibody-CCP complex, or measuring the amount of theCCP not separated from the anti-CCP antibody-CCP complex. The measuringmay be implemented by detecting a signal from the detectable makerattached to the CCP. Separating of the CCP may be performed bycentrifugation, precipitation, salting out, dialysis, filtration,chromatography, electrophoresis, or any combination thereof.Chromatography may include substitution chromatography, size-exclusionchromatography, ion-exchange chromatography, or any combination thereof.The measuring of the level of the CCP and/or the anti-CCP antibody-CCPcomplex may be performed by western blotting, enzyme linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA),radioimmunodiffusion, Ouchterlony radioimmunodiffusion (ODD), rocketimmunoelectrophoresis, tissue immunostaining, immunoprecipitation assay,complement fixation assay, fluorescence-activated cell sorting (FACS),protein chip, spectrometry, spectroscopy, or any combination thereof.

For example, the measuring of the level of the CCP, anti-CCP antibody,or anti-CCP antibody-CCP complex may be performed by ELISA. ELISA mayinclude any of various types of ELISA, including a direct ELISA using alabeled antibody to detect an antigen fixed to a solid support, anindirect ELISA using a labeled (secondary) antibody to detect a capture(primary) antibody in an antigen-antibody complex in which the capture(primary) antibody is bound to an antigen fixed to a solid support, adirect sandwich ELISA using a labeled antibody to detect the antigen ofan antibody-antigen complex bound to a solid support, and an indirectsandwich ELISA using a labeled secondary antibody to detect an antibodywhich is bound to the antigen of an antibody-antigen complex bound to asolid support.

For example, when sandwich ELISA is used, the sandwich ELISA may includethe following steps: for example, coating the CCP as a primary antibodyon a surface of a solid support; contacting a sample taken from asubject and the primary antibody to induce an antigen-antibody reaction;reacting a resulting product from the inducing of antigen-antibodyreaction with a secondary antibody bound with an enzyme; and detectingthe activity of the enzyme.

The solid support may be formed of a hydrocarbon polymer (for example,polystyrene or polypropylene), glass, metal, or gel. For example, thesolid support may be a microtiter plate. The secondary antibody boundwith an enzyme may include an enzyme that may catalyze a color reaction,fluorescent reaction, luminescent reaction, or infrared reaction, forexample, alkaline phosphatase, β-galactosidase, horseradish peroxidase,luciferase, or cytochrome P450. For example, when the enzyme bound tothe secondary antibody is alkaline phosphatase, a substrate for thisenzyme bound to the secondary antibody may be a substrate for colorreaction, for example, a substrate including bromochloroindolylphosphate (BCIP), nitro blue tetrazolium (NBT),naphthol—AS-B1-phosphate, and enhanced chemifluorescence (ECF). When theenzyme bound to the secondary antibody is horseradish peroxidase, asubstrate for this enzyme bound to the secondary antibody may bechloronaphthol, aminoethyl carbazole, diaminobenzidine, D-luciferin,lucigenin (bis-N-methyl acridinium nitrate), resolupin benzyl ether,luminol, an amplex red reagent (10-acetyl-3,7-dihydroxyphenoxazine), HYR(p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine),ABTS (2,2′-azino-bis[3-ethylbenzothiazoline-6-sulphonic acid]),o-phenylenediamine (OPD), naphthol/pie Ronin, glucose oxidase, t-NBT(nitroblue tetrazolium) or m-PMS (phenzaine methosulfate).

In some embodiments, the anti-CCP antibody detection method may furtherinclude determining the subject to be at a high risk for rheumatoidarthritis if the level of the CCP, anti-CCP antibody, and/or anti-CCPantibody-CCP complex is higher than a control group sample. The controlgroup sample may be a sample obtained from a non-rheumatoid arthritispatient or a subject with a low risk for rheumatoid arthritis.

The determining may include: comparing the amount of the anti-CCPantibody in the sample taken from the subject with the amount of theanti-CCP antibody in the control group sample; and determining thesubject as a rheumatoid arthritis patient or to be at a high risk forrheumatoid arthritis if the amount of the anti-CCP antibody is higherthan the amount of the anti-CCP antibody in the control group sample.

The anti-CCP antibody detection method may further include measuring thepresence, amount, and/or level of at least one different rheumatoidarthritis markers, or measuring the level of a material thatspecifically bind to the at least one different rheumatoid arthritismarkers or a fragment thereof. The different rheumatoid arthritismarkers may be a RF, an anti-CCP antibody, a CRP, an ESR or acombination thereof.

The terms and elements that are referred to herein in conjunction withthe anti-CCP antibody detection method according to any of theabove-described embodiments and overlap with those used to describe theclaimed rheumatoid arthritis diagnosis compositions or kits have thesame meanings as used with regard to the claimed rheumatoid arthritisdiagnosis compositions or kits.

According to another aspect of the present disclosure, there is provideda method of screening a diagnostic marker for rheumatoid arthritis, themethod including: modifying an amino acid sequence in a CCP; contactingthe CCP having the modified amino acid sequence with a test sample takenfrom a rheumatoid arthritis patient to form an anti-CCP antibody-CCPcomplex; and measuring the level of the anti-CCP antibody.

The modifying of the amino acid sequence may include addition, deletion,substitution, and/or post-translational modification of an amino acid.

The addition of an amino acid may be adding an amino acid in the middleof sequence, a N-terminal, a C-terminal, or both N-terminal andC-terminal of the CCP. For example, an amino acid to be added may bec-aminocaproic acid.

The substitution of an amino acid may be substituting at least one ofthe amino acids present at a N-terminal, a C-terminal, or between theN-terminal and C-terminal. The substitution of an amino acid may besubstituting an alkaline, acidic, polar, hydrophobic, aromatic, orpost-translationally modified amino acid by an amino acid having adifferent characteristic therefrom.

The modifying of the amino acid sequence may be modifying a sequence ofat least one amino acid present between citrulline of the CCP anddisulfide bond-forming cysteine in the C-terminal direction.

The screening method may further include contacting the CCP having themodified amino acid sequence with a test sample taken from a rheumatoidarthritis patient and with a control group sample.

The screening method may include determining the CCP as a candidatediagnostic marker for rheumatoid arthritis if the level of the anti-CCPantibody detected in the test sample taken from a rheumatoid arthritispatient is higher than the level of the anti-CCP antibody detected inthe control group sample.

A number of ring-forming amino acids in the CCP is not limited as longas the CCP can bind to an anti-CCP antibody. For example, the number ofring-forming amino acids in the CCP may be, for example, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25.

The screening method may further include measuring the sensitivityand/or specificity of anti-CCP antibodies for the diagnosis ofrheumatoid arthritis based on the measured level of the anti-CCPantibody. The screening method may further include selecting a cut-offlevel of anti-CCP antibodies with an optical sensitivity and/orspecificity based on a receiver operating characteristic (ROC) curve.

A ROC curve as a plot of the relation between sensitivity andspecificity in a specific diagnostic test may represent the diagnosticaccuracy of a specific diagnostic model. The ROC may be a graphgenerated by plotting sensitivity of all possible cut-off points for aspecific diagnostic model on Y-axis against 1-specificity on X-axis. Thespecific diagnostic model may be a diagnostic model for rheumatoidarthritis in which a specific level of anti-CCP antibody is determinedas a cut-off level for positivity with disease (abnormal) and/ornegativity without disease (normal patients). The larger the area underthe ROC curve (AUC), the higher the accuracy of the determineddiagnostic model. With the assumption that a total area of the ROC curveis 1, a diagnostic model may be determined as having high accuracy whenthe AUC is 0.5 or greater, 0.6 or greater, 0.7 or greater, 0.8 orgreater, or 0.9 or greater. A diagnostic model may be determined as ahigher accuracy when it is closer to the upper left corner of the ROC.

The term “sensitivity” may refer to the probability that an individualwith the disease will test positive in a specific diagnostic model.

The term “specificity” may refer to the probability that a disease-freeindividual will test negative in a specific diagnostic model.

The screening method may further include measuring the sensitivitiesand/or specificities of CCPs with modified amino acid sequences andselecting a CCP with higher sensitivity and/or specificity for thediagnosis of rheumatoid arthritis than the other CCPs with modifiedamino acid sequences.

The terms and elements referred to herein in conjunction with a methodof screening a diagnostic marker for RA according to any of theabove-described embodiments and overlap with those used to describe theclaimed rheumatoid arthritis diagnosis compositions or kits, or anti-CCPantibody detection methods are understood to have the same meanings asused with regard to the claimed rheumatoid arthritis diagnosiscompositions or kits, or anti-CCP antibody detection methods.

One or more embodiments of the present disclosure will now be describedin detail with reference to the following examples. However, theseexamples are only for illustrative purposes and are not intended tolimit the scope of the one or more embodiments of the presentdisclosure.

Example 1 Preparation of Peptides and Patient Serum Samples and AssayMethods Thereof

The reagents and devices used were as follows.

Phosphate buffered saline (PBS, Cat. No: LB201-02, available fromWelGene, Daejeon, Korea), Tween-20 (Cat. No: 274348) for washing, bovineserum albumin (BSA, Cat. No: A1253, available from Sigma-aldrich, St.Louis, USA), HRP-conjugated anti-human IgG antibody (Cat. No: ab6858,available from Abcam, Cambridge, UK) as a secondary antibody foranti-CCP antibody detection were purchased. A 96-well microplate (Cat.No: 439454) and a TMB substrate (Cat. No: 34021) were purchased fromThermo (Rockford, USA). Peptides were synthesized by Peptron (Daejon,Korea). The results from enzyme linked immunosorbent assay (ELISA) wereanalyzed using a microplate reader (available from Bio-rad, Hercules,USA).

Table 1 shows sequence information of the used peptides, including usualand unusual amino acids such as citrulline and aminocaproic acid.

TABLE 1 SEQ ID Sequence No. Peptide information  1 CCP HCHQESTXGRSRGCG 2 RF EGLHNHY  3 HSH15 HSHQESTXGRSRGSG  4 ZZH17 ZZHSHQESTXGRSRGSG  5HSH17 HSHQESTXGRSRGSGZZ  6 ZZH19 ZZHSHQESTXGRSRGSGZZ  7 CCP10PHCHQESTXGPSRGCG  8 CCP11A HCHQESTXGRARGCG  9 CCP12P HCHQESTXGRSPGCG 10CCPPAP HCHQESTXGPAPGCG * X indicates citrulline, and Z indicatesaminocaproic acid. * Peptides with SEQ ID NOS. 1, and 7 to 10 may be incyclic form due to cysteines 2 and 14 forming disulfide bond.

Sandwich enzyme linked immunosorbent assay (ELISA) using the peptides ofTable 1 were performed on the samples from 4 test groups. Serum samplesof RA patients (30 subjects), OA patients (15 subjects), SLE patients(25 subjects), and control group samples (25 normal subjects) wereprepared.

TABLE 2 No. of Gender Type of disease patients Age (Female/Male)Rheumatoid arthritis (RA) 30 51.21 (25-74) 22/8 Osteoarthritis (OA) 1547.27 (20-75) 13/2 Systemic lupus erythemathode 25 38.32 (22-83) 24/1(SLE) Normal control group 25 52.56 (29-76)  15/10

Each of the prepared peptides of Table 1 was diluted with PBS to about10 μg/ml, added by about 100 μl to a microplate, and then coated atabout 4° C. overnight, followed by adding about 200 μl of BSA solution(10 mg/mL) to each well of the peptide coated microplate to block atroom temperature for about 1 hour, and washing the microplate with a PBSincluding about 0.1% of Tween-20 three times to use the microplate it inthis assay. The patient samples and normal control group sample werediluted by 200 times and added triplet by about 100 μl to the wells ofthe peptide-coated microplate. After being cultured at room temperaturefor about 1 hour, the resulting microplate was washed three times in thesame manner as the above-described washing. To detect autoimmuneantibodies in the samples, 100 μl of an anti-human IgG antibody solution(500 nm/mL) was added to each cell of the microplate and incubated atroom temperature for about 1 hour, followed by washing three times inthe same manner as the above-described washing. After 100 μl of a TMBsubstrate solution was added thereto and reacted at room temperature forabout 30 minutes, finally 100 μl of a 2M sulfuric acid solution wasadded as a stop solution to terminate the reaction, followed byabsorbance measurement at a wavelength of about 450 nm using amicroplate reader.

Example 2 Identification of Binding Site of CCP to Antibody by UsingModified Peptide Obtained by Adding Aminocaproic Acid to Terminal of CCP

Autoimmune antibody levels in the blood samples of normal patients andvarious types of arthritis patients were analyzed by enzyme linkedimmunosorbent assay (ELISA) using the peptides of SEQ ID NOS. 1 to 6shown in Table 1.

Aminocaproic acid may specifically bind to a polystyrene resinsubstrate. This characteristics of aminocaproic acid may be utilized toidentify major binding sites in the peptide sequence of CCP forautoimmune antibodies present in the bloods of a RA patient. Forexample, a modified CCP may be prepared by substituting disulfidebond-forming cysteine in CCP with serine to obtain a linearlized CCPwithout disulfide bond and adding aminocaproic acid to one or both of N-and C-terminals of the linearlized CCP to have a structure with N-or/and C-terminals able to bind to the microplate. If a modified CCPwith immobilized N-terminal and free C-terminal presents higher bindingaffinity than that of a modified CCP with immobilized C-terminal andfree N-terminal, the C-terminal of CCP may be identified as a bindingsite with high binding strength to autoimmune antibody of CCP.

(1) Identification of Role of CCP's Cyclic Structure in Binding to CCP'sAutoimmune Antibody

FIGS. 1A, 1B, 1C, 1D, 1E, and 1F are graphs illustrating the levels ofautoimmune antibody in the blood samples of the normal patients andvarious types of arthritis patients, wherein X-axis denotes samples andY-axis denotes absorbance. The results of FIGS. 1A, 1B, 1C, 1D, 1E, and1F are the results of assay using CCP, RF, HSH15, ZZH17, HSH17, andZZH19, respectively, wherein HSH15 is a modified peptide obtained bysubstituting disulfide bond-forming cysteine in CCP with serine toeliminate disulfide bond, ZZH17 is a modified peptide obtained by addingaminocaproic acid to N-terminal of HSH15, HSH17 is a modified peptideobtained by adding aminocaproic acid to C-terminal of HSH15, and ZZH19is a modified peptide obtained by adding aminocaproic acid to N- andC-terminals of HSH15.

The average absorbance in each sample calculated based on the results ofFIGS. 1A to 1F, and statistical significances (p-value, %) with respectto normal patients calculated using t-test are shown in Table 3.

TABLE 3 RA OA SLE Normal Average Average Average Average Peptide(absorbance) P value (absorbance) P value (absorbance) P value(absorbance) CCP 0.2522 <0.0001 0.2314 <0.0001 0.2347 <0.0001 0.0874 RF0.2534 0.0002 0.2791 0.0003 0.3417 <0.0001 0.1484 HSH15 0.1942 <0.00010.2277 0.0001 0.2704 <0.0001 0.1067 ZZH17 0.2667 <0.0001 0.2344 <0.00010.2738 <0.0001 0.1102 HSH17 0.2681 <0.0001 0.2916 <0.0001 0.3569 <0.00010.1412 ZZH19 0.4008 <0.0001 0.3069 <0.0001 0.3693 <0.0001 0.1609

The absorbance values of all the arthritis patient samples analyzedusing the various peptides were statistically significantly higher thanthose of the normal control group samples. ZZH19 is a modified peptidethat exposes citrulline moiety like CCP when coated on a microplate. theRA patient sample treated with ZZH19 was found to represent an averageabsorbance of about 0.4008, which was higher than an average absorbanceof about 0.3069 in the OA patient sample and an average absorbance ofabout 0.3693 in the SLE patient sample. ZZH19 of the other modified CCPsincluding aminocaproic acid was also found to have a highest averageabsorbance in the RA patient samples, indicating that a cyclic structurewith exposed citrulline may play a significant role in the diagnosis ofRA.

(2) Identification of Role of C-Terminal of CCP in Binding to CCP'sAutoimmune Antibody

Referring to Table 3, HSH17 with exposed N-terminal represents anaverage absorbance of about 0.2681 in the RA patients, which was lowerthan an average absorbance of about 0.2916 in the OA patients, and about0.3569 in the SLE patients. Meanwhile, ZZH17 with exposed C-terminal wasfound to represent an average absorbance of about 0.2667 in the RApatient samples, which was higher than an average absorbance of about0.2344 in the OA patient samples.

FIGS. 2A and 2B are graphs of sensitivity and specificity, respectively,of the peptides treated in the samples of various types of arthritispatients, calculated based on the ROC curves plotted using the resultsof Table 3. According to an autoimmune antibody detection assay usingthe CCP, the CCP was found to have a sensitivity of about 93.3%, about93.3%, and about 100% in the RA, OA, and SLE patient samples,respectively, and was found to have a specificity of about 88.5, about88.5, and about 84.6 in the RA, OA, and SLE patient samples,respectively. RF was found to have a lower sensitivity and lowerspecificity than CCP. HSH17 was found to have a highest sensitivity ofabout 100% in all of the RA, OA, and SLE patient samples, while theaminocaproic acid-added modified peptides, including HSH17, were foundto have a lower specificity than CCP in the RA patient samples. HSH17,although was highest insensitivity, was found to have a relatively lowspecificity of about 69.2%, 73.1%, and 69.2% in the RA, OA, and SLEpatient samples, respectively. ZZH19 immobilized on a microplate in asimilar pattern as CCP and ZZH17 with N-terminal immobilized on amicroplate were found to have a relatively high specificity of about84.6% and about 80.8%, respectively, when treated in the RA patientsamples. Therefore, it was identified that the C-terminal domain ofamino acid sequence of CCP may play an important role in the binding ofautoimmune antibodies.

Example 3 Identification of Autoimmune Antibody Level in ArthritisPatients Detected Using C-Terminal Sequence-Modified CCP and StatisticalSignificance Test Thereon

Autoimmune antibody levels in the blood samples of normal patients andvarious arthritis patients were analyzed by ELISA using the peptides ofSEQ ID NOS. 7 to 10 shown in Table 1.

In particular, novel candidate diagnostic markers for rheumatoidarthritis were prepared by modifying a hydrophilic amino acid sequencein the C-terminal domain of CCP identified in Example 2 into ahydrophobic amino acid. The 10th amino acid arginine and 12th amino acidproline of CCP in the C-terminal direction from the 8th amino acidcitrulline of CCP were substituted by proline to construct “CCP10P” and“CCP12P”, respectively. The 11th amino acid serine was substituted byalanine to construct “CCP11A”, and the 10^(th), 11^(th), and 12^(th)amino acids were substituted by proline, alanine, and proline,respectively, to construct “CCPPAP”. The autoimmune antibody detectionability of these constructed peptides in various types of arthritispatients were analyzed.

FIGS. 3A, 3B, 3C, 3D, and 3E are graphs illustrating the levels ofautoimmune antibody in the blood samples of the normal patients andvarious types of arthritis patients, detected using the peptides of SEQID NOS. 7 to 10 of Table 1. The average absorbance in each samplecalculated based on the results of FIGS. 3A to 3E and statisticalsignificances (p-value, %) with respect to normal patients calculatedusing t-test are shown in Table 4.

TABLE 4 RA OA SLE Normal Average Average Average Average Peptide(absorbance) P value (absorbance) P value (absorbance) P value(absorbance) CCP 0.2522 <0.0001 0.2314 <0.0001 0.2347 <0.0001 0.0874CCP10P 0.1870 <0.0001 0.1875 0.0002 0.2777 <0.0001 0.0994 CCP11A 0.3091<0.0001 0.2689 <0.0001 0.3321 <0.0001 0.1125 CCP12P 0.2445 <0.00010.2847 <0.0001 0.3619 <0.0001 0.1250 CCPPAP 0.2549 <0.0001 0.2591<0.0001 0.3287 <0.0001 0.1140

The absorbance values of all the arthritis patient samples analyzedusing the various peptides were statistically significantly higher thanthose of the normal control group samples. CCP10P was found to representan average absorbance of about 0.1870 in the RA patients, which waslower than an average absorbance of about 0.1875 in the OA patients andan average absorbance of about 0.2777 in the SLE patients. CCP12P andCCPPAP were both found to represent a lower average absorbance in the RApatients than in the OA and SLE patients. However, CCP11A was found tohave an average absorbance of about 0.3094 in the RA patients, which washigher than an average absorbance of about 0.2689 in the OA patients.

CCP11A, CCP12P, and CCPPAP were found to have a high specificity ofabout 100% with respect to the RA, and in particular, CCP11A withsubstituted 11th amino acid had about 11.5% higher specificity than CCP.However, CCP10P with substituted 10th amino acid was found to have aspecificity of about 76.9%, which was about 11.6% lower than CCP,indicating that the 10th amino acid arginine of CCP is an importantsequence in the diagnosis of rheumatoid arthritis.

FIGS. 4A, 4B, and 4C show ROC curves of CCP, and CCP10P and CCP11A asmodified CCPs including substituted amino acid, respectively, in RApatient samples with respect to normal patient samples. FIGS. 4D, 4E,and 4F show ROC curves of CCP, and CCP10P and CCP11A, respectively, inRA patient samples with respect to OA patient samples. AUCs, positiveprediction rates (PPR, %), and negative prediction rates (NPR, %)obtained from the ROC curves of FIGS. 4A to 4F are shown in Table 5.

TABLE 5 Control Sensitivity Specificity PPR NPR Peptide group (%) (%)AUC (%) (%) CCP Normal 93.3 88.5 0.9718 93.3 88 OA 70.0 60.0 0.6133 7060 CCP10P Normal 83.3 76.9 0.8370 83.3 76 OA 30.0 93.3 0.5030 30 93.3CCP11A Normal 86.7 100.0 0.9756 86.7 100 OA 63.3 80.0 0.6930 63.3 80

Referring to Table 5, CCP11A had an AUC of about 0.9756 in RA patientswith respect to normal patient samples, which was larger than that ofCCP by about 0.0038, and an AUC of about 0.6930 with respect to OApatient samples, which was larger than a CCP's AUC of about 0.6133 byabout 0.0797. Specificity and NPR, which are known as the core factorsthat determine the accuracy or reliability of a diagnostic assay, CCP11Awere higher than those of CCP in the detection of RA with respect toboth normal and OA.

FIGS. 5A and 5B are graphs illustrating sensitivity and specificity ofCCP, CCP10P, and CCP11A, and statistical significances thereof in thediagnosis for RA with respect to normal (FIG. 5A) and OA patient samples(FIG. 5B) as controls.

The statistical significances were calculated using the McNemer test.The McNemer test is a test on a 2×2 table to calculate a differencebetween paired two tests and a statistical significance level. Referringto FIG. 5A, a p-value between the results of the two assays with CCP andCCP10P in the RA patient samples with respect to normal patient samplesas control group was 1, and a difference therebetween was 0.0%,indicating that the results of the two assays with CCP and CCP10P arestatistically very similar. Referring to FIG. 5B, a p-value between theresults of the two assays with CCP and CCP10P in the RA patient sampleswith respect to OA patient samples as control group was about 0.004, anda difference therebetween was about 37.78%. A p-value between theresults of the two assays with CCP and CCP11A in the RA patient sampleswith respect to OA patient samples was about 0.04, and a differencetherebetween was about 26.67%. Therefore, the results of the assay withCCP10P had a statistically significantly difference from the assayresults with CCP and CCP11A.

As described above, according to the one or more of the aboveembodiments of the present invention, a CCP may be used to efficientlydiagnose rheumatoid arthritis. A rheumatoid arthritis diagnosiscomposition or kit (including the CCPs) may be used to efficientlydiagnose rheumatoid arthritis. A method of detecting an anti-CCPantibody in a subject may efficiently provide information required inthe diagnosis for rheumatoid arthritis. An efficient method of screeningnovel diagnostic markers for rheumatoid arthritis is provided.

It should be understood that the exemplary embodiments described thereinshould be considered in a descriptive sense only and not for purposes oflimitation. Descriptions of features or aspects within each embodimentshould typically be considered as available for other similar featuresor aspects in other embodiments.

While one or more embodiments of the present invention have beendescribed with reference to the figures, it will be understood by thoseof ordinary skill in the art that various changes in form and detailsmay be made therein without departing from the spirit and scope of thepresent invention as defined by the following claims.

What is claimed is:
 1. A peptide having an amino acid sequence with asequence identity of about 80% or more to at least one of amino acidsequences of SEQ ID NOS: 3 to
 10. 2. The peptide of claim 1, wherein thepeptide is detectably labeled.
 3. A rheumatoid arthritis diagnosiscomposition comprising a peptide having an amino acid sequence with asequence identity of about 80% or more to at least one of amino acidsequences of SEQ ID NOs: 3 to
 10. 4. The rheumatoid arthritis diagnosiscomposition of claim 3, further comprising a material that specificallybinds to at least one rheumatoid arthritis marker or a fragment thereof.5. The rheumatoid arthritis diagnosis composition of claim 4, whereinthe at least one rheumatoid arthritis marker is a rheumatoid factor(RF), an anti-cyclic citrullinated peptide (anti-CCP) antibody, aC-reactive protein (CRP), or a combination thereof.
 6. A method ofdetecting an anti-cyclic citrullinated peptide (anti-CCP) antibody, themethod comprising: forming an anti-CCP antibody-peptide complex bycontacting a sample taken from a subject and a peptide having an aminoacid sequence with a sequence identity of about 80% or more to at leastone of amino acid sequences of SEQ ID NOs: 3 to 10 to bind ananti-cyclic citrullinated peptide (CCP) antibody present in the sampleto the peptide; and detecting a level of the anti-CCP antibody-peptidecomplex in the sample.
 7. The method of claim 6, further comprisingdetermining that the subject is at high risk for rheumatoid arthritis ifthe level of the anti-CCP antibody-peptide complex in the sample ishigher than a level of anti-CCP antibody-peptide complex in a controlgroup sample.
 8. The method of claim 6, further comprising measuring alevel of at least one different rheumatoid arthritis marker in thesample.
 9. The method of claim 8, wherein the measuring of the level ofat least one different rheumatoid arthritis marker comprises measuring alevel of a rheumatoid factor (RF), an anti-CCP antibody, or a C-reactiveprotein (CRP) or measuring the erythrocyte sedimentation rate (ESR). 10.The method of claim 6, wherein the detecting of the level of theanti-CCP antibody-peptide complex is performed using western blotting,enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA),radioimmunodiffusion, Ouchterlony radioimmunodiffusion (ODD), rocketimmunoelectrophoresis, tissue immunostaining, immunoprecipitation assay,complement fixation assay fluorescence-activated cell sorting (FACS),protein chip, or a combination thereof.
 11. A method of screening adiagnostic marker for rheumatoid arthritis, the method comprising:modifying at least one amino acid of a cyclic citrullinated peptide(CCP) present between citrulline and disulfide bond-forming cycteine ina C-terminal direction from the citrulline; forming anti-CCPantibody-peptide complexes by bringing the modified CCP to contact atest sample taken from a rheumatoid arthritis patient and a controlgroup sample to bind the modified CCP to anti-CCP antibodies present inthe test sample and the control sample group; measuring levels of theanti-CCP antibodies in the anti-CCP antibody-peptide complexes in thetest sample and the control group sample; and determining the CCP as acandidate diagnostic marker for rheumatoid arthritis if the level of theanti-CCP antibody in the anti-CCP antibody-peptide complex in the testsample is higher than that in the control group sample.
 12. The methodof claim 11, wherein a number of amino acids forming a cycle in the CCPis in the range of about 5 to about
 25. 13. The method of claim 11,further comprising, before the forming of the anti-CCP antibody-peptidecomplexes, substituting at least one hydrophilic amino acid between thecitrulline of the CCP and the disulfide bond-forming cycteine in theC-terminal direction with a hydrophobic amino acid.
 14. The method ofclaim 11, further comprising screening a CCP having an area under theROC curve (AUC) of about 0.7 or greater, wherein the ROC curve is areceiver operating characteristic curve of sensitivity on Y-axis against1-specificity on X-axis, the ROC curve being obtained by plottingsensitivity against 1-specificity using all the levels of anti-CCPantibodies in samples as cut-off points for diagnosing rheumatoidarthritis.
 15. The method of claim 11, further comprising screening aCCP having a sensitivity and/or specificity of about 0.7 or greater.